Insulators are regulatory DNA sequences which form gene boundaries and protect genes from the influence of neighboring chromatin. Insulator-proteins bind to insulator DNA elements in the genome and tether those regions to subnuclear structures thus forming chromatin loops such that enhancer/silencer elements of one gene may not gain access to the promoter of a neighboring gene. Given the complexity of the human genome, one would expect that a variety of insulators and insulator- proteins regulate transcription of the estimated 35,000 genes. For example, in Drosophila alone, four types of insulators and binding proteins have been reported. In contrast, only one type of insulator (chicken globin HS4) and insulator-protein (CTCF) has been reported so far in vertebrates. CTCF mediated insulator activity is modulated by methylation of DNA. We have characterized a CpG-free, CTCF-independent vertebrate insulator (SP-10 insulator) from a testis-specific gene. The SP-10 insulator is conserved between mice and men. Preliminary mutation studies have indicated that transcription factor YY1 may mediate the SP-10 insulator function. In the proposed pilot study, we will investigate if YY1 interacts with the SP-10 insulator in vitro (using EMSA) and in a physiological context (using ChIP) (Specific Aim 1);and address the requirement of YY1 for SP-10 insulator function using RNAi in a cell culture model as well as a YY1 conditional knockout mouse model (Specific Aim 2). This feasibility study is a small self-contained research project with the potential to lead to a larger hypothesis-driven study. A recent study has shown that YY1 is essential for spermatogenesis. The current study will expand the role of YY1 to testis-specific gene transcription and in the long-term to male infertility. PUBLIC HEALTH RELEVANCE: Incidence of infertility is on the rise;it has been estimated that in the United States one in six couples in the reproductive age group are unable to conceive. The male factor contributes to 50% of the infertility cases. Treatment options are limited for men owing to a poor understanding of the mechanisms controlling sperm production (spermatogenesis). Here we propose to study the role of a transcription factor YY1 in regulating testis-specific gene transcription. Since the mouse model has recently shown that absence of YY1 in male germ cells leads to defective spermatogenesis, the proposed studies on the function of YY1 in testis are highly important for male infertility and are therefore of high public health relevance.